CN114196583B - Streptomyces thermophilus, synergistic biological agent containing same and application thereof - Google Patents
Streptomyces thermophilus, synergistic biological agent containing same and application thereof Download PDFInfo
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- CN114196583B CN114196583B CN202111534692.4A CN202111534692A CN114196583B CN 114196583 B CN114196583 B CN 114196583B CN 202111534692 A CN202111534692 A CN 202111534692A CN 114196583 B CN114196583 B CN 114196583B
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- streptomyces
- carbon monoxide
- thermophilic
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05D—INORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C; FERTILISERS PRODUCING CARBON DIOXIDE
- C05D1/00—Fertilisers containing potassium
-
- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05G—MIXTURES OF FERTILISERS COVERED INDIVIDUALLY BY DIFFERENT SUBCLASSES OF CLASS C05; MIXTURES OF ONE OR MORE FERTILISERS WITH MATERIALS NOT HAVING A SPECIFIC FERTILISING ACTIVITY, e.g. PESTICIDES, SOIL-CONDITIONERS, WETTING AGENTS; FERTILISERS CHARACTERISED BY THEIR FORM
- C05G3/00—Mixtures of one or more fertilisers with additives not having a specially fertilising activity
-
- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05G—MIXTURES OF FERTILISERS COVERED INDIVIDUALLY BY DIFFERENT SUBCLASSES OF CLASS C05; MIXTURES OF ONE OR MORE FERTILISERS WITH MATERIALS NOT HAVING A SPECIFIC FERTILISING ACTIVITY, e.g. PESTICIDES, SOIL-CONDITIONERS, WETTING AGENTS; FERTILISERS CHARACTERISED BY THEIR FORM
- C05G3/00—Mixtures of one or more fertilisers with additives not having a specially fertilising activity
- C05G3/60—Biocides or preservatives, e.g. disinfectants, pesticides or herbicides; Pest repellants or attractants
-
- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05G—MIXTURES OF FERTILISERS COVERED INDIVIDUALLY BY DIFFERENT SUBCLASSES OF CLASS C05; MIXTURES OF ONE OR MORE FERTILISERS WITH MATERIALS NOT HAVING A SPECIFIC FERTILISING ACTIVITY, e.g. PESTICIDES, SOIL-CONDITIONERS, WETTING AGENTS; FERTILISERS CHARACTERISED BY THEIR FORM
- C05G3/00—Mixtures of one or more fertilisers with additives not having a specially fertilising activity
- C05G3/80—Soil conditioners
-
- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05G—MIXTURES OF FERTILISERS COVERED INDIVIDUALLY BY DIFFERENT SUBCLASSES OF CLASS C05; MIXTURES OF ONE OR MORE FERTILISERS WITH MATERIALS NOT HAVING A SPECIFIC FERTILISING ACTIVITY, e.g. PESTICIDES, SOIL-CONDITIONERS, WETTING AGENTS; FERTILISERS CHARACTERISED BY THEIR FORM
- C05G5/00—Fertilisers characterised by their form
- C05G5/10—Solid or semi-solid fertilisers, e.g. powders
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W30/00—Technologies for solid waste management
- Y02W30/40—Bio-organic fraction processing; Production of fertilisers from the organic fraction of waste or refuse
Abstract
The invention relates to a thermophilic streptomyces carbon monoxide and a synergistic biological microbial agent containing the same, which utilize the biological efficacy of the thermophilic streptomyces carbon monoxide and combine the functional characteristics of a microbial synergistic agent, so that the microorganism has stronger self-nitrogen fixation capacity, potassium and trace element decomposition capacity, siderophore production capacity and cellulase capacity, and physiological diseases caused by elements such as potassium deficiency, calcium, iron, magnesium, sulfur and the like of plants are prevented; and is capable of synthesizing more plant growth promoting signal molecules, such as: volatile gas components such as indoleacetic acid and acetoin promote plant growth, and increase biomass indexes such as plant height, root weight, stem thickness, leaf area and the like; meanwhile, micromolecular organic acid components such as acetic acid secreted by the thermophilic streptomyces carbon monoxide are used for eliminating soil hardening, loosening soil, neutralizing alkalinity and reducing soil saline-alkali diseases; more bacteriostatic substances such as streptomycin are secreted, so that the growth and reproduction of harmful bacteria are more effectively inhibited, the soil environment is purified, and the root systems of crops are protected. The synergistic biological agent comprises but is not limited to microbial agents, bacterial fertilizers, biopesticides, biological fertilizers, plant growth promoters, antibacterial agents, preservatives, disinfectants or food preservatives, and is used as an auxiliary agent when being independently applied or being prepared or applied to microbial agents, bacterial fertilizers, biological organic fertilizer products and derivative products thereof.
Description
Technical Field
The invention belongs to the field of agricultural microorganisms, relates to a biological bacterial fertilizer technology, and in particular relates to a thermophilic streptomyces carbon monoxide and a synergistic biological microbial agent containing the same and application thereof.
Background
The biological bacterial fertilizer is an emerging important fertilizer in the facility agriculture production in China, and the popularization and the application of the biological bacterial fertilizer are not only the extension of scientific formula fertilization, but also important components for developing ecological and organic facility agriculture. The biological bacterial fertilizer in the prior art generally has the problem of unstable bacterial activity, the bacterial activity depends on the biological efficacy of living microorganisms, and the activity of exogenous living microbial preparations is influenced by a plurality of factors, such as soil microenvironment, physicochemical properties, indigenous microorganisms, root secretions, application modes and the like, so that the biological bacterial fertilizer taking microorganisms as main materials cannot stably exert the biological efficacy, further influence the growth promotion of crops, and the research and development of the biological bacterial fertilizer with synergistic effect has important application value.
Disclosure of Invention
The invention aims to overcome the defects of the prior art, provides the thermophilic streptomyces carbon monoxide and the synergistic biological microbial agent containing the thermophilic streptomyces carbon monoxide, and utilizes the biological efficacy of the thermophilic streptomyces carbon monoxide and combines the functional characteristics of the microbial synergistic agent, so that the microorganism has stronger autonomous nitrogen fixation capability, potassium and trace element decomposition capability, siderophore production capability and cellulase capability, and can prevent physiological diseases caused by elements such as potassium deficiency, calcium, iron, magnesium, sulfur and the like of plants; and is capable of synthesizing more plant growth promoting signal molecules, such as: volatile gas components such as indoleacetic acid and acetoin promote plant growth, and increase biomass indexes such as plant height, root weight, stem thickness, leaf area and the like; meanwhile, small molecular organic acid components such as acetic acid secreted by the thermophilic streptomyces carbon monoxide are used for eliminating soil hardening, loosening soil, neutralizing alkalinity and reducing soil saline-alkali diseases; more bacteriostatic substances such as streptomycin are secreted, so that the growth and reproduction of harmful bacteria are more effectively inhibited, the soil environment is purified, and the root systems of crops are protected.
The invention solves the technical problems by adopting the following technical scheme:
a strain of streptomyces thermophilus, said strain being named: fs-1, class designation: thermophilic carbon monoxide streptomycete (Streptomyces thermocarboxydus) with a preservation number of CGMCC No.21628 and a preservation date of: 2021, 1-15, deposit unit: china general microbiological culture Collection center, preservation address: no. 1 and No. 3 of the north cinquefoil of the morning sun area of beijing city.
The synergistic microbial inoculum containing the thermophilic streptomyces carbon monoxide comprises the following components in parts by mass: 10-50 parts of thermophilic carbon monoxide streptomyces Fs-1 bacterial powder, 5-10 parts of diatomite, 5-30 parts of kaolin, 5-10 parts of light calcium carbonate, 1-5 parts of PEG80001, 1-10 parts of humic acid, 1-10 parts of plant ash and 1-10 parts of microbial synergist.
Moreover, the preparation process of the streptomyces thermophilus Fs-1 bacterial powder comprises the following steps:
(1) And (3) strain expansion culture: the activated streptomyces thermophilus Fs-1 is inoculated into a culture medium of Gao's No. 1, and is cultured for 2 days under the conditions of 28 ℃ and 180r/min to obtain a streptomyces thermophilus Fs-1 seed solution, wherein the characteristic value of the number of viable bacteria of the streptomyces thermophilus seed solution is 1.0x10 9 -6.0×10 9 cfu/mL；
(2) Preparation of pretreatment mixture: taking the following components in parts by weight: 0.01-10 parts of rice, 0.01-12 parts of soluble starch, 0.01-6 parts of wheat bran, 0.01-2 parts of malt extract powder, 0.01-1 part of yeast extract powder, 0.01-10 parts of peptone, 0.01-2 parts of cottonseed meal powder, 0.01-5 parts of soybean meal powder, 0.01-0.4 part of magnesium sulfate, 0.01-1 part of ammonium sulfate, 0.01-1 part of dipotassium hydrogen phosphate, 0.01-1 part of potassium nitrate, 0.01-0.5 part of cobalt chloride and 0.01-0.5 part of sodium chloride, adding water to adjust the water content to 10-40 percent, and the solid content=3: 7w/w, sterilizing at 121deg.C for 30min, and cooling;
(3) And (3) preparation of a fermentation product: taking the pretreatment mixture prepared in the step (2), inoculating the seed liquid of the streptomyces thermophilus Fs-1 prepared in the step (1) according to the inoculation amount of 10% by mass, uniformly stirring, fermenting for 14 days at the temperature of 28 ℃ under the ventilation condition, and periodically supplementing sterile water and turning over the materials during the fermentation to obtain the solid fermentation product of the streptomyces thermophilus Fs-1;
(4) Preparing bacterial powder: and (3) drying the fermented product prepared in the step (3) at a low temperature by using a fluidized bed dryer, and superfine grinding the fermented product to 400-2000 meshes to obtain the thermophilic streptomyces carbon monoxide Fs-1 bacterial powder with high viable count, wherein the characteristic value of the viable count is 2-200 hundred million/g.
The microbial synergist comprises the following components in parts by weight: the plant or microorganism cell wall component factors comprise 0.01-5 parts of plant or microorganism cell wall component factors, 0.001-0.01 part of environment stress metal ions and 0.001-0.05 part of microorganism signal sensing molecules, the balance of the plant or microorganism cell wall component factors comprise one or more of glucose, fructose, galactose, arabinose, xylose, rhamnose, fucose, mannose, uronic acid, chitin, chitosan oligosaccharide, dextran, chitosan oligosaccharide and N-acetylglucosamine, the environment stress metal ions comprise one or more of zinc sulfate, ferrous sulfate, cobalt chloride and lanthanum chloride, and the microorganism signal sensing molecules comprise gamma-butyrolactone or N-lipo homoserine lactone.
The preparation method of the microbial synergist comprises the following steps: at room temperature of 20 ℃, adding plant or microorganism cell wall component factors into an acetic acid aqueous solution with the mass concentration of 0-0.1%, wherein the adding ratio g: mL = 50-100:850, performing ultrasonic treatment for 20min under the ultrasonic frequency of 20-40kHz to prepare colorless or yellowish mixed solution A; mixing and dissolving environment stress metal ions and microorganism signal sensing molecules in water, and adding the following components in proportion of mg: mL: mL = 30-60mg:1-10mL:100mL of mixed solution A is added under the stirring state, after the mixed solution A is completely dissolved, 1mol/LNaOH is selected to adjust the pH value of the mixed solution to 5.0-9.0, then purified water is used for quantifying the mixed solution to 1L, insoluble matters are removed through filtration, and the finished product mother solution of the regulator is obtained, and a spray drying device is selected, wherein the temperature of an air inlet is 150-180 ℃ and the temperature of an air outlet is 70-80 ℃ under the low temperature condition, so as to prepare the finished product powder of the regulator.
The application of a synergistic microbial agent containing thermophilic streptomyces carbon monoxide is provided: can be used alone or as auxiliary preparation when microbial agent, bacterial fertilizer and bio-organic fertilizer are prepared or applied. The individual administration included: (1) the powder state is used as a base fertilizer, and the using method comprises the following steps: seed dressing or mixed application with soil, chemical fertilizer, farmyard manure and the like, and the total amount of the seed dressing is recommended to be 200-800g per mu of land; (2) the mother solution diluted aqueous solution is used as topdressing, and the using method comprises the following steps: root irrigation or spraying, and diluting the mother liquor 5-10 times, wherein the total amount of the mother liquor is recommended to be 2.5-9.0L per mu.
The invention has the advantages and positive effects that:
1. the thermophilic streptomyces carbon monoxide strain is separated from the rhizosphere area of tomatoes, is one of dominant rhizosphere microorganisms of plants such as tomatoes, and has good re-colonization capability in rhizosphere of plants such as tomatoes. Because the streptomyces thermophilus strain has the characteristics of high temperature resistance, cellulase production and the like, the streptomyces thermophilus strain is usually used for high temperature composting, and the streptomyces thermophilus strain Fs-1 disclosed by the invention also has the capabilities of autonomously fixing nitrogen, dissolving potassium and producing siderophores and acetic acid, and is beneficial to eliminating soil hardening, loosening soil, neutralizing alkalinity and reducing saline-alkali diseases of soil. Meanwhile, the strain can metabolize volatile gas components such as indoleacetic acid, acetoin and the like, so that the thermophilic streptomyces carbon monoxide Fs-1 strain has the capability of promoting the growth of crops, namely, biomass indexes such as the height, root weight, stem thickness, leaf area and the like of the crops are increased, the root growth and root activity of the crops are promoted, and the strain can be newly developed into microbial agents or microbial fertilizers. In addition, each capability of the strain is tested to have space for optimization and re-promotion.
2. The streptomyces thermophilus synergistic biological agent of the invention can synthesize more plant growth promoting signal molecules than single bacterial strains, for example: volatile gas components such as indoleacetic acid and acetoin can promote plant growth more effectively. Meanwhile, compared with the single strain, the synergistic biological microbial inoculum provided by the invention can secrete more organic acid, so that soil hardening, soil loosening, alkalinity neutralization and soil saline-alkali disease reduction can be effectively eliminated. Finally, the synergistic biological inoculant can secrete more antibacterial substances such as streptomycin, inhibit growth and reproduction of harmful bacteria, purify the soil environment and protect crop root systems.
3. The synergistic biological inoculant of the streptomyces thermophilus disclosed by the invention comprises, but is not limited to, a microbial inoculant, a bacterial fertilizer, a biological pesticide, a biological fertilizer, a plant growth promoting agent, an antibacterial agent, a preservative, a disinfectant or a food preservative, and can be independently applied or used as an auxiliary preparation when the microbial inoculant, the bacterial fertilizer, a biological organic fertilizer product and derivative products thereof are prepared or applied.
4. Microbial synergists in the present invention include, but are not limited to, streptomyces carbophilus Fs-1, and microorganisms of the classes Cyanobacter, proteobacteria, actinobactylobacteria and Planocomycetes, which are capable of increasing the colonization and survival rate of Cyanobacter, proteobacteria, actinobactylobacteria and Planocytes-gate microorganisms in the rhizosphere, and increasing the relative abundance and ratio of these microorganisms.
5. In the preparation process of the microbial synergist, the problems of solubility of different components in water, inactivation or discoloration of substances such as monosaccharide and the like at a high temperature are fully considered, and the preparation process of ultrasonic treatment, acid-soluble alkali adjustment, ionic polymerization, low Wen Chuigan and the like is adopted, so that the mutual interference among the components is reduced to the greatest extent, and the biological activity of the components is reserved. Finally mixing with the thermophilic streptomyces carbon monoxide Fs-1 bacterial powder, and ensuring that the content of each component, especially the content of the synergist in the product is more accurate.
6. The microbial synergist provided by the invention can effectively colonise the thermophilic streptomyces carbon monoxide on the plant rhizosphere surface and exert the ecological effect, and provides the following assistance: (1) providing energy sources required by growth of microorganisms in a colonization window period, (2) providing microbial plant interaction signal molecules, starting mutual identification between the microorganisms and the plants, establishing a reciprocal, symbiotic and reciprocal relationship, providing microbial metabolism regulation signal molecules, and (3) regulating and controlling antibacterial substance synthesis pathways such as tryptophan-auxin metabolic pathway, pyruvic acid-acetic acid-butanediol pathway and streptomycin regulated by transcription factor DasR in the thermophilic streptomyces carbon monoxide Fs-1.
Drawings
FIG. 1 is a colony morphology of Streptomyces thermophilus Fs-1 according to the invention;
FIG. 2 is a phylogenetic tree diagram of the Streptomyces thermophilus Fs-1 of the present invention;
FIG. 3 is a graph showing the effect of Streptomyces carbothermophilus on the growth of corn, wheat and tomato in the examples of the invention;
FIG. 4 shows the effect of different concentrations of Streptomyces carbothermophilus Fs-1 on Arabidopsis growth in the examples of the present invention;
FIG. 5 shows the effect of S.thermophilus Fs-1 on autofixing nitrogen, decomposing potassium and producing cellulase in the present invention;
FIG. 6 shows the effect of the mixed synergistic bacteria on synthesizing auxin in the embodiment of the invention;
FIG. 7 is a graph of tomato rhizosphere microorganism horizontal colony abundance before and after treatment with the potentiator of the present invention;
FIG. 8 is a graph showing comparison of the metabolic flows of the rhizosphere microorganisms KEGG of tomatoes before and after the treatment with the potentiator of the present invention;
Detailed Description
The invention is further illustrated by the following examples, which are intended to be illustrative only and not limiting in any way.
1. The invention firstly separates a strain of thermophilic streptomyces carboxidans from tomato rhizosphere soil, and the name of the strain is: fs-1, class designation: the preservation number of the thermophilic streptomyces carbon monoxide Streptomyces thermocarboxydus is CGMCC No.21628, and the preservation date is as follows: 2021, 1-15, deposit unit: the China general microbiological culture Collection center, north Chen West Lu No. 1, 3, of the Korean region of Beijing city, is specifically shown by the annex preservation certificate.
Identification of strains of the thermophilic carbon monoxide chain, 16S RNA sequence information for the Fs-1 strain is as follows:
ttcccggggggggtgcttaccatgcagtcgaacgatgaagcccttcggggtggattagtggcgaacgggtgagtaacacgtgggcaatctgccctgcactctgggacaagccctggaaacggggtctaataccggatactgatcgccttgggcatccttggtgatcgaaagctccggcggtgcaggatgagcccgcggcctatcagcttgttggtgaggtaatggctcaccaaggcgacgacgggtagccggcctgagagggcgaccggccacactgggactgagacacggcccagactcctacgggaggcagcagtggggaatattgcacaatgggcgaaagcctgatgcagcgacgccgcgtgagggatgacggccttcgggttgtaaacctctttcagcagggaagaagcgaaagtgacggtacctgcagaagaagcgccggctaactacgtgccagcagccgCGGTAATACGTAGGGCGCGAGCGTTGTCCGGAATTATTGGGCGTAAAGAGCTCGTAGGCGGCTTGTCGCGTCGGTTGTGAAAGCCCGGGGCTTAACCCCGGGTCTGCAGTCGATACGGGCAGGCTAGAGTTCGGTAGGGGAGATCGGAATTCCTGGTGTAGCGGTGAAATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGGATCTCTGGGCCGATACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGGTGGGCACTAGGTGTGGGCGACATTCCACGTCGTCCGTGCCGCAGCTAACGCATTAAGTGCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGTGGCTTAATTCGACGCAACGCGAAGAACCTTACCAAGGCTTGACATACACCGGAAACGTCCAGAGATGGGCGCCCCCTTgtggtcggtgtacaggtggtgcatggctgtcgtcagctcgtgtcgtgagatgttgggttaagtcccgcaacgagcgcaacccttgtcccgtgttgccagcaggcccttgtggtgctggggactcacgggagaccgccggggtcaactcggaggaaggtggggacgacgtcaagtcatcatgccccttatgtcttgggctgcacacgtgctacaatggccggtacaatgagctgcgataccgcgaggtggagcgaatctcaaaaagccggtctcagttcggattggggtctgcaactcgaccccatgaagtcggagtcgctagtaatcgcagatcagcattgctgcggtgaatacgttcccgggccttgtacacaccgcccgtcacgtcacgaaagtcggtaacacccgaagccggtggcccaacccccttgtggggagggagcgtcgaagtgaaccggcctt。
the strain has the following culture characteristics:
the strain was cultured in liquid media such as Gao's first medium, LB medium, YEME medium, TSBY medium, starch, YMS medium, ISP2 medium, etc., and the growth and pigment production of the strain were observed. The thallus particles are bigger and can generate spores.
As shown in FIG. 1, on the culture medium of Gaoshi No. 1, the thermophilic carbon monoxide streptomycete strain grows well, the colony is slightly raised, the texture is compact and dry, the strain is in a micro-velvet shape and is opaque, aerial hyphae are white or off-white, basal hyphae are light yellow, the growth is limited, and pigment is generated. The phylogenetic tree diagram of the thermophilic streptomyces carbon monoxide Fs-1 is shown in figure 2.
2. The strain is separated and cultured to prepare the streptomyces thermophilus Fs-1 bacterial powder, and the preparation process comprises the following steps:
(1) And (3) strain expansion culture: inoculating activated Streptomyces thermophilus Fs-1 into culture medium of Khaki No. 1, and culturing at 28deg.C and 180r/min for 2 days to obtain seed solution of Streptomyces thermophilus Fs-1 with number of viable bacteria of characteristic value of 1.0X10 9 -6.0×10 9 cfu/mL；
(2) Preparation of pretreatment mixture: taking rice, soluble starch, wheat bran, malt extract powder, yeast extract powder, peptone, cotton seed cake powder, soybean meal powder, magnesium sulfate, ammonium sulfate, dipotassium hydrogen phosphate, potassium nitrate, cobalt chloride and sodium chloride, and uniformly mixing the following components in parts by mass: 0.01-10 parts of rice, 0.01-12 parts of soluble starch, 0.01-6 parts of wheat bran, 0.01-2 parts of malt extract powder, 0.01-1 part of yeast extract powder, 0.01-10 parts of peptone, 0.01-2 parts of cottonseed meal powder, 0.01-5 parts of soybean meal powder, 0.01-0.4 part of magnesium sulfate, 0.01-1 part of ammonium sulfate, 0.01-1 part of dipotassium hydrogen phosphate, 0.01-1 part of potassium nitrate, 0.01-0.5 part of cobalt chloride and 0.01-0.5 part of sodium chloride, adding water to adjust the water content to 10-40% (water: solid=3:7w/w), sterilizing at 121 ℃ for 30min, and cooling for later use;
(3) And (3) preparation of a fermentation product: taking the pretreatment mixture prepared in the step (2), inoculating the seed liquid of the streptomyces thermophilus Fs-1 prepared in the step (1) according to the inoculation amount of 10% by mass, uniformly stirring, fermenting for 14 days at the temperature of 28 ℃ under the ventilation condition, and periodically supplementing sterile water and turning over the materials during the fermentation to obtain the solid fermentation product of the streptomyces thermophilus Fs-1;
(4) Preparing bacterial powder: and (3) drying the fermented product prepared in the step (3) at a low temperature by using a fluidized bed dryer, and superfine grinding the fermented product to 400-2000 meshes to obtain the thermophilic streptomyces carbon monoxide Fs-1 bacterial powder with high viable count, wherein the characteristic value of the viable count is 2-200 hundred million/g.
3. The synergistic microbial inoculum containing the thermophilic streptomyces carbon monoxide is prepared by the following components in parts by mass: 10-50 parts of thermophilic carbon monoxide streptomyces Fs-1 bacterial powder, 5-10 parts of diatomite, 5-30 parts of kaolin, 5-10 parts of light calcium carbonate, 80001-10 parts of PEG, 1-5 parts of humic acid, 1-10 parts of plant ash and 0.01-10 parts of microbial synergist.
Specifically, the microbial synergist comprises the following components in parts by weight: 0.01-5 parts of plant or microorganism cell wall component factors, 0.001-0.01 part of environmental stress metal ions and 0.001-0.05 part of microorganism signal sensing molecules, and the balance of water.
The plant or microorganism cell wall component factors comprise one or more of glucose, fructose, galactose, arabinose, xylose, rhamnose, fucose, mannose, uronic acid, chitin, chitosan oligosaccharide and N-acetylglucosamine, the environment stress metal ions comprise one or more of zinc sulfate, ferrous sulfate, cobalt chloride and lanthanum chloride, and the microorganism signal sensing molecules comprise gamma-butyrolactone or N-lipoylhomoserine lactone.
4. The preparation method of the microbial synergist in the synergist containing the streptomyces thermophilus comprises the following steps: at room temperature of 20 ℃, adding plant or microorganism cell wall component factors into an acetic acid aqueous solution with the mass concentration of 0-0.1%, wherein the adding ratio g: mL = 50-100:850, performing ultrasonic treatment for 20min under the ultrasonic frequency of 20-40kHz to prepare colorless or yellowish mixed solution A; mixing and dissolving environment stress metal ions and microorganism signal sensing molecules in water, and adding the following components in proportion of mg: mL: mL = 30-60mg:1-10mL:100mL of mixed solution A is added under the stirring state, after the mixed solution A is completely dissolved, 1mol/LNaOH is selected to adjust the pH value of the mixed solution to 5.0-9.0, then purified water is used for quantifying the mixed solution to 1L, insoluble matters are removed through filtration, and the finished product mother solution of the regulator is obtained, and a spray drying device is selected, wherein the temperature of an air inlet is 150-180 ℃ and the temperature of an air outlet is 70-80 ℃ under the low temperature condition, so as to prepare the finished product powder of the regulator.
The experimental method and experimental result of the embodiment of the invention are as follows:
the growth effect promotion test is carried out on potted wheat, corn and tomatoes in the embodiment of the invention, and the growth effect is shown in figure 3:
soaking the crop seeds in clear water for 24 hours, uniformly spotting the crop seeds in nutrient soil, covering a layer of thin soil on the surface layer, watering, sprouting for 5 days, starting to perform different treatments, adding 5mL of tap water into a CK group, adding 5mL of thermophilic streptomyces carbon monoxide bacterial suspension into a treatment group, performing irrigation treatment every 2 days, and observing and recording the growth conditions of wheat, corn and tomatoes.
Plant height: the plant height was measured every 2d, and the total length of wheat and corn was measured with the ground soil surface as a reference point.
Fresh weight, dry weight, root length: after 14d of culture, randomly selecting a certain amount of wheat, corn and tomato roots from the treatment group and the CK group, pulling up the roots, cleaning soil at the roots, wiping the roots with gauze, weighing fresh amount of the roots, and measuring root length of the roots. And after the measurement is finished, drying at 60 ℃ for 1.2 hours to determine the dry weight of the material.
Corn stalk is thick and 1/2 leaf width: after 14d of wheat and corn growth, the treatment group stopped watering the inoculum and, when grown to 30d, the corn was measured for stem thickness and leaf width at 1/2 of the total leaf length.
Chlorophyll content determination: 1.0g of leaf sample is weighed and added into a mortar, a small amount of quartz sand, calcium carbonate powder and 2-3 ml of 80% acetone solution are added, the mixture is ground into homogenate, and 10ml of 80% acetone solution is added for continuous grinding until the tissue becomes white. Standing for 3-5 min for extraction. The extract was filtered with filter paper into a 50ml brown volumetric flask, rinsed with acetone solution until the filter paper and residue were green-free. The volume was fixed to 50ml with 80% acetone, shaken well, OD663, OD645 were measured, and the formula was calculated: ρa= 12.72OD663-2.59OD645; ρb= 22.88OD645-4.67OD663; total chlorophyll ρt=ρa+ρb=20.29od645+8.05od663.
The test results are shown in the following table:
TABLE 1 effect of Streptomyces thermophilus on corn and wheat growth results
TABLE 2 influence of Streptomyces thermophilus on tomato growth
As shown in FIG. 3, the experimental results show that the thermophilic carbon monoxide Streptomyces Fs-1 has a promoting effect on the growth of corn, wheat and tomatoes. After the thermophilic streptomyces carboxidans Fs-1 is applied, plant height and root weight indexes of corn, wheat and tomatoes are obviously increased, and biomass indexes such as stem thickness, leaf area and the like of the corn are also obviously improved.
At the same time, the embodiment also makes a test of the growth effect (Arabidopsis thaliana) of the potted plant promotion mode
Washing seeds of Arabidopsis thaliana with sterile water for several times, then spotting the seeds on a sterile flat plate paved with sterile filter paper as uniformly as possible by a pipetting gun, sealing a sealing film, then carrying out vernalization for 2d at 4 ℃, then carrying out germination in a culture box at 22 ℃, spotting the germinated Arabidopsis thaliana into nutrient soil after germination, culturing in the culture box, and carrying out regular watering.
Total leaf area, total fresh weight of arabidopsis thaliana. Transplanting the arabidopsis sprouted in the culture dish into nutrient soil, after the arabidopsis is grown normally for 15 days, selecting the arabidopsis with consistent size for experiment, preparing bacterial suspensions with different concentrations according to a full factor experiment table, adding 1ml of each bacterial suspension, then co-culturing for 7 days, photographing the arabidopsis seedlings every day (controlling the same magnification and the same mobile phone), measuring leaf area by using imageJ software, and measuring the weight of overground parts.
As shown in FIG. 4, the experimental result shows that the thermophilic carbon monoxide streptomyces Fs-1 has an effect of promoting the growth of leaves of arabidopsis, and the promotion capacity has a single-peak curve relationship between the application concentration and the interval. The low concentration (1%) has the most obvious promotion effect.
Then, tests on the capability of the thermophilic streptomyces carbon monoxide Fs-1 to autonomously fix nitrogen, dissolve potassium, produce siderophores and cellulase are carried out,
as shown in FIG. 5, on the selection medium of the Aphis Bei Modan, the strain of the Streptomyces thermophilus Fs-1 grows well, and transparent rings can be produced on the culture medium of the potassium feldspar, so that the strain has the self-nitrogen fixation capacity and the potassium dissolving capacity. Meanwhile, the value of the activity unit of the siderophore is 0.253 by using a 630nmCAS detection method, and the siderophore production capacity is higher. The cellulose-producing capacity of the culture medium is detected by using a cellulose Congo red culture medium method, and the value of a cellulase transparent circle (H/C) is found to be 3.22.
The effect of the mixed synergistic microbial agent of the thermophilic carbon monoxide streptomyces Fs-1 is shown in figure 6.
The method for measuring the indoleacetic acid comprises the following steps: accurately weighing 1mL of the sample, adding 1mL of pre-frozen 80% glacial methanol, fully and uniformly stirring, centrifuging at 4 ℃ for 10min at 5000r/min, and discarding the precipitate to obtain a sample extract containing indoleacetic acid. The color developing agent needs to be prepared and used at present, and the preparation method comprises the following steps: 0.5mol/L FeCl 2 (to be stored in dark place) 0.15mL, concentrated sulfuric acid 3mL and distilled water 5 mL. The ELISA plate was added with 40uL of the indoleacetic acid extract, and 160uL of the color development solution was added to each of the indoleacetic acid extract and the blank with distilled water having the same volume as that of the sample extract. After mixing uniformly, shading and color development reaction is carried out for 30min at constant temperature, and the absorbance value of the sample at 530uL is measured by a spectrophotometer. The content of indoleacetic acid in the sample (ug/g) = (a×v1)/(w×v2), wherein a is the concentration of indoleacetic acid in the mycelium extract that participates in the color reaction, ug/mL. V1 is the total volume of the extract in the sample, mL. W is the weight of the sample, g. V2 is the volume of the sample extract taking part in the color reaction, mL.
The test results of the mixed synergistic agent of the thermophilic carbon monoxide streptomyces Fs-1 for promoting the synthesis of volatile gases are as follows:
GC/MS analysis of volatile material components, the headspace solid-phase microextraction combined with gas chromatography-mass spectrometry (GC-MS) is used to collect and analyze VOCs produced during microbial fermentation. Collection of volatile materials: taking 5ml of fermentation liquor sample in a 20ml headspace sample injection bottle, placing the sample in a constant-temperature water bath on a magnetic stirrer at 45 ℃ and 1200rpm for 30 minutes, inserting an aged 75 mu CAR/PDMS solid-phase microextraction fiber head, adsorbing the sample on the headspace for 30 minutes, enriching volatile substances on the extraction head, and directly injecting the volatile substances into a gas chromatography-mass spectrometer for analysis.
Gas chromatography conditions. Chromatographic column model: VF-5MS (30 m. Times.0.25 mm. Times.0.25 μm); sample inlet temperature: 250 ℃; carrier gas: he, purity 99.999%; split ratio: 10:1, a step of; flow rate: 1mL/min; programming temperature: the initial temperature is 40 ℃, kept for 3min, heated to 150 ℃ at 4 ℃/min, kept for 1min, heated to 250 ℃ at 8 ℃/min, and kept for 6min. Mass spectrometry conditions: mass separator: an ion trap; ion source: EI; ionization energy: 70eV; ion trap temperature: 220 ℃; transmission line temperature: 280 ℃; solvent delay time: 1.5min; a full scanning mode; scanning range: 43-500m/z; searching a spectrum library: NIST11. The mass spectrum of the sample component was compared with a standard chart, and the relative content of each component was determined using 2-methyl-3-heptanone (CAS: 13019-20-0) as an internal standard.
The obtained result shows that the combination of different synergists can obviously enhance the synthesis of 3-hydroxy-2-butanone, 2, 3-butanediol, ethyl acetate, 2, 3-butanedione and other substances by the thermophilic streptomyces carbon monoxide Fs-1. Wherein, 3-hydroxy-2-butanone and 2, 3-butanediol belong to microorganism source organic volatile matters for promoting plant growth. In the synergistic microbial inoculum combination 1, the content of 3-hydroxy-2-butanone reaches 0.0538, which is higher than that of a control group by more times. In the synergistic microbial agent combination 3, the content of the 2, 3-butanediol reaches 0.0008, and compared with the result that the control group and other synergistic microbial agents are lower than the detection limit, the synergistic microbial agent combination has obvious increase.
TABLE 3 Synthesis of Mixed synergistic microbial Agents with plant growth gas promoting Capacity
The microbial synergist increases the synthesis of bacteriostatic substances such as streptomycin and the number of probiotics on the surface of the rhizosphere, and the results are as follows:
taking compound synergists with different concentrations as experimental test objects, and setting a natural growth group and a synergist experimental group (group one to group four) in an experiment; each group was provided with a bacterial control group and a sterile control group. Each experimental group was set up with 6 groups in parallel.
And (3) plant experiment operation, namely placing tomato seedlings into an intelligent artificial climate illumination incubator for culturing for one week so as to adapt to an experiment environment. After the adaptation period is over, abnormal tomato seedlings are removed, and the rest tomato seedlings are randomly grouped, wherein 6 tomato seedlings are arranged in each group. The aseptic group is subjected to aseptic treatment. Sterilizing the soil mixture at 121 ℃ for 20 minutes; the tomato seedlings were sterilized with 75% ethanol for 1 minute, then rinsed 3 times with sterile distilled water, and the irrigation was performed with sterile distilled water. The bacteria group does not need any treatment, and tap water is used for irrigation.
Soil flora samples were collected, after 21 days, and field soil, rhizosphere soil, and intra-root soil were isolated as described below. Discarding the soil layer with the thickness of 1cm at the top in the basin, and shoveling the mixed soil with the thickness of 1g into a centrifuge tube subjected to sterilization treatment to collect a field soil sample; shaking the plant root system to remove loose soil until only soil within 1 mm from the root system surface remains, and collecting the root system surface soil as a rhizosphere soil sample; the root system was rinsed with 75% ethanol, after which the root system was placed in a clean and sterile 250mL conical tube containing 50mL of sterile distilled water. Placed on a shaker and shake the root system at 180rpm for 20 minutes 3 times. The resulting turbid solution was collected and centrifuged twice to form a compact precipitate, which was collected as an in-root soil sample. All soil samples in the fields, rhizosphere and root were frozen fresh and stored at-80 ℃ for future use.
As a result, as shown in FIG. 7, at the gate classification level, the total number of bacterial gates of < 0.01% was classified into other, and the experimental group yielded 12 bacterial gates, namely, cyanobacteria (Cyanobacter), proteus (Proteus), actinobacillus (Actinobacillus), fungium (Planocytes), acidobacter (Acidobacter), agrimonia (Gemmatimonades), bacteroides (Bacteroides), lloydia viridis (Chroflexi), parcuacterium, verrucomicrobia (Verrucomicrobia), helicobacter (Saccharacteria), and Chlamydia (Chlamydia) in order from high to low, with the relative abundance of 0.01% as the threshold. The relative abundance ratio of Proteus (Proteus), thick-walled bacteria (Phylum Firmicutes), bacteroides (Bacteroides) and helicobacter (Saccharobacter) was increased in the experimental group to which the synergist was added, and the relative abundance increase in rhizosphere soil was significant. One of the synergistic agent components enhances the colonization ability and survival proportion of microorganisms of the genus Cyanobacteria, proteobacteria, actionobacteria and Plactomycetes on the rhizosphere surface of tomatoes, and improves the relative abundance and the ratio of microorganisms of the genus Cyanobacteria.
Meanwhile, in rhizosphere soil, the addition of exogenous potentiators significantly increased the relative abundance of pyrimidine metabolism (Pyrimidine metabolism), peptidoglycan biosynthesis (Peptidogycan biosynthesis), nucleotide excision repair (Nucleotide excision repair), protein repair (Protein repair), and streptomycin biosynthesis (Streptomycin biosynthesis) (fig. 8).
Although embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that: various substitutions, changes and modifications are possible without departing from the spirit and scope of the invention and the appended claims, and therefore the scope of the invention is not limited to the disclosure of the embodiments.
Sequence listing
<110> university of Tianjin science and technology
<120> a thermophilic Streptomyces carbophilus, a synergistic biological agent containing the same and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1433
<212> DNA
<213> Fs-1 Strain 16S RNA sequence (Unknown)
<400> 1
ttcccggggg gggtgcttac catgcagtcg aacgatgaag cccttcgggg tggattagtg 60
gcgaacgggt gagtaacacg tgggcaatct gccctgcact ctgggacaag ccctggaaac 120
ggggtctaat accggatact gatcgccttg ggcatccttg gtgatcgaaa gctccggcgg 180
tgcaggatga gcccgcggcc tatcagcttg ttggtgaggt aatggctcac caaggcgacg 240
acgggtagcc ggcctgagag ggcgaccggc cacactggga ctgagacacg gcccagactc 300
ctacgggagg cagcagtggg gaatattgca caatgggcga aagcctgatg cagcgacgcc 360
gcgtgaggga tgacggcctt cgggttgtaa acctctttca gcagggaaga agcgaaagtg 420
acggtacctg cagaagaagc gccggctaac tacgtgccag cagccgcggt aatacgtagg 480
gcgcgagcgt tgtccggaat tattgggcgt aaagagctcg taggcggctt gtcgcgtcgg 540
ttgtgaaagc ccggggctta accccgggtc tgcagtcgat acgggcaggc tagagttcgg 600
taggggagat cggaattcct ggtgtagcgg tgaaatgcgc agatatcagg aggaacaccg 660
gtggcgaagg cggatctctg ggccgatact gacgctgagg agcgaaagcg tggggagcga 720
acaggattag ataccctggt agtccacgcc gtaaacggtg ggcactaggt gtgggcgaca 780
ttccacgtcg tccgtgccgc agctaacgca ttaagtgccc cgcctgggga gtacggccgc 840
aaggctaaaa ctcaaaggaa ttgacggggg cccgcacaag cggcggagca tgtggcttaa 900
ttcgacgcaa cgcgaagaac cttaccaagg cttgacatac accggaaacg tccagagatg 960
ggcgccccct tgtggtcggt gtacaggtgg tgcatggctg tcgtcagctc gtgtcgtgag 1020
atgttgggtt aagtcccgca acgagcgcaa cccttgtccc gtgttgccag caggcccttg 1080
tggtgctggg gactcacggg agaccgccgg ggtcaactcg gaggaaggtg gggacgacgt 1140
caagtcatca tgccccttat gtcttgggct gcacacgtgc tacaatggcc ggtacaatga 1200
gctgcgatac cgcgaggtgg agcgaatctc aaaaagccgg tctcagttcg gattggggtc 1260
tgcaactcga ccccatgaag tcggagtcgc tagtaatcgc agatcagcat tgctgcggtg 1320
aatacgttcc cgggccttgt acacaccgcc cgtcacgtca cgaaagtcgg taacacccga 1380
agccggtggc ccaaccccct tgtggggagg gagcgtcgaa gtgaaccggc ctt 1433
Claims (5)
1. A strain of streptomyces thermophilus carbon monoxide, characterized in that: the name of the strain is: fs-1, class designation: thermophilic carbon monoxide streptomycete (Streptomyces thermocarboxydus) with a preservation number of CGMCC No.21628 and a preservation date of: 2021, 1-15, deposit unit: china general microbiological culture Collection center, preservation address: no. 1 and No. 3 of the north cinquefoil of the morning sun area of beijing city.
2. A synergistic microbial agent containing thermophilic carbon monoxide streptomycete is characterized in that: comprising a strain of Streptomyces thermophilus according to claim 1.
3. A synergistic microbial agent containing thermophilic carbon monoxide streptomycete is characterized in that: comprises the following components in parts by weight: 10-50 parts of thermophilic carbon monoxide streptomyces Fs-1 bacterial powder, 5-10 parts of diatomite, 5-30 parts of kaolin, 5-10 parts of light calcium carbonate, 80001-10 parts of PEG, 1-5 parts of humic acid and 1-10 parts of plant ash, wherein the name of the strain is: fs-1, class designation: thermophilic carbon monoxide streptomycete (Streptomyces thermocarboxydus) with a preservation number of CGMCC No.21628 and a preservation date of: 2021, 1-15, deposit unit: china general microbiological culture Collection center, preservation address: no. 1 and No. 3 of the north cinquefoil of the morning sun area of beijing city.
4. A synergistic agent containing streptomyces thermophilus as claimed in claim 3, wherein: the preparation process of the streptomyces thermophilus Fs-1 bacterial powder comprises the following steps:
(1) And (3) strain expansion culture: the activated streptomyces thermophilus Fs-1 is inoculated into a culture medium of Gao's No. 1, and is cultured for 2 days under the conditions of 28 ℃ and 180r/min to obtain a streptomyces thermophilus Fs-1 seed solution;
(2) Preparation of pretreatment mixture: taking the following components in parts by weight: 0.01-10 parts of rice, 0.01-12 parts of soluble starch, 0.01-6 parts of wheat bran, 0.01-2 parts of malt extract powder, 0.01-1 part of yeast extract powder, 0.01-10 parts of peptone, 0.01-2 parts of cottonseed meal powder, 0.01-5 parts of soybean meal powder, 0.01-0.4 part of magnesium sulfate, 0.01-1 part of ammonium sulfate, 0.01-1 part of dipotassium hydrogen phosphate, 0.01-1 part of potassium nitrate, 0.01-0.5 part of cobalt chloride and 0.01-0.5 part of sodium chloride, adding water to adjust the water content to 10-40%, sterilizing at 121 ℃ for 30min, and cooling for later use;
(3) And (3) preparation of a fermentation product: taking the pretreatment mixture prepared in the step (2), inoculating the seed liquid of the streptomyces thermophilus Fs-1 prepared in the step (1) according to the inoculation amount of 10% by mass, uniformly stirring, fermenting for 14 days at the temperature of 28 ℃ under the ventilation condition, and periodically supplementing sterile water and turning over the materials during the fermentation to obtain the solid fermentation product of the streptomyces thermophilus Fs-1;
(4) Preparing bacterial powder: and (3) drying the fermented product prepared in the step (3) at a low temperature by using a fluidized bed dryer, and superfine grinding the fermented product to 400-2000 meshes to obtain the thermophilic streptomyces carbon monoxide Fs-1 bacterial powder with high viable count, wherein the characteristic value of the viable count is 2-200 hundred million/g.
5. The biological agent containing the thermophilic streptomyces carbon monoxide according to claim 4, which is characterized in that: the number characteristic value of the living bacteria of the thermophilic carbon monoxide streptomycete seed liquid in the step (1) is 1.0 multiplied by 10 9 -6.0×10 9 cfu/mL, water in step (2):
solids = 3:7w/w.
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| CN104988095A (en) * | 2015-07-03 | 2015-10-21 | 北京农学院 | Thermophilic carbon monoxide streptomycete low-temperature subspecies Dstr3-3 and application thereof |
| CN110982748A (en) * | 2019-12-26 | 2020-04-10 | 北京林业大学 | Preparation and application of compound microbial agent for coal gangue artificial ecological matrix |
| CN113564065A (en) * | 2020-10-14 | 2021-10-29 | 辽宁省微生物科学研究院 | Streptomyces fradiae G-1 and application thereof |
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| CN103484396A (en) * | 2013-08-15 | 2014-01-01 | 北京农学院 | New strain of streptomyces thermocarboxydus and application thereof |
| CN104988095A (en) * | 2015-07-03 | 2015-10-21 | 北京农学院 | Thermophilic carbon monoxide streptomycete low-temperature subspecies Dstr3-3 and application thereof |
| CN110982748A (en) * | 2019-12-26 | 2020-04-10 | 北京林业大学 | Preparation and application of compound microbial agent for coal gangue artificial ecological matrix |
| CN113564065A (en) * | 2020-10-14 | 2021-10-29 | 辽宁省微生物科学研究院 | Streptomyces fradiae G-1 and application thereof |
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